Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium

Abstract

The proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1β expression and release are tightly controlled by changes in intracellular Ca²⁺ ([Ca²⁺]_i), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells following treatment with the proinflammatory cytokine TNF-α. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-κB-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1β. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1β expression was not altered after direct ATP stimulation in human umbilical vein endothelial cells. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca²⁺ diffusion into the cell to regulate IL-1β. High-throughput flow cytometric analysis demonstrated that TNF-α treatments lead to elevated [Ca²⁺]_i, corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-α-associated increases in [Ca²⁺]_i, blocked phosphorylation of the NF-κB-p65 protein, and reduced IL-1β transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca²⁺]_i regulation via the Panx1 channel induces a feed-forward effect on NF-κB to regulate IL-1β synthesis and release in endothelium during inflammation.